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White J, Meier J. Colony to colorimetry in 6 h: Oi, oi, oi, gurias! Free Download e-Books Yes, those batches will open and close very quickly because they re so small. Metacyclic trypomastigotes were prepared as previously described [ 48 ].
Recombinant protein expression The T. Immunoblotting and immunofluorescence Immunoblotting and immunofluorescence were performed as previously described [ 51 ].
TcDHH1 47 kDa was used as a control of the metacyclogenesis western blotting assay. Polysome sedimentation profiles Polysomes were separated on a sucrose density gradient as previously described, performed once for each condition [ 53 ].
The drugs used here were puromycin, which causes premature chain termination during translation, thus disassembling polysomes [ 54 ]; and cycloheximide, that interferes with the translocation step in protein synthesis, thus blocking eukaryotic translational elongation and stabilizing polysomes [ 54 ].
The resulting fractions were analyzed by western blotting using rabbit anti-protein A , dilution for DRBD2 detection and mouse anti-S7 dilution , kindly provided by Dr. S7 25 kDa is a small ribosomal protein commonly used as a control. The fractionation was performed twice for each treatment and condition. Immunoprecipitation assays Immunoprecipitation IP assays were performed with cytoplasmic extracts of exponentially growing epimastigotes.
The equivalent of 5. Approximately 5. Shotgun proteomic data acquisition and analysis Digested extracts of the immunoprecipitated lysates were fractionated by a NanoLC Easy system Thermo Fisher Scientific.
Sequences of T. PatternLab for proteomics 4.
The results were postprocessed to accept PSM with less than 5 ppm from the average ppm of the pre-validated results. The T.